Porcine reproductive and respiratory syndrome (PRRS), often characterized by late-term abortions and still births in sows and respiratory disease in nursery pigs, has resulted in extensive economic losses in the swine industry for over a decade. (16). First described in 1987 in the United States as “mystery swine disease”, it spread rapidly, being reported in Europe in 1990 and subsequently across the world.
Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, is a small, enveloped, positive-stranded RNA virus consisting of eight overlapping open reading frames (ORFs). (19, 21). The virus is genetically, antigenically, and pathogenically heterogenic. (16). Substantial sequence divergence exists between the European and North American genotypes of the virus. (1, 9, 11-16, 20-21). Within each genotype, the PRRSV genomic sequences also vary significantly. (8, 13, 14, 20).
Modified live-attenuated vaccines (MLV) are currently used for the protection against PRRS mainly by providing protection against clinical disease. (18, 24). These modified live-attenuated vaccines, such as Ingelvac® PRRS MLV, have reduced the incidence and severity of PRRS outbreaks on many farms. A severe form of PRRS, designated acute or atypical PRRS, has recently been reported in the Midwestern United States. Many of these acute outbreaks occurred in PRRSV MLV-vaccinated herds, suggesting that the commonly used modified live-attenuated vaccines are not fully effective. The occurrence of the acute syndrome in vaccinated pigs resulted in the introduction of another MLV, Ingelvac® PRRS ATP, to the market in February 2000.
Other concerns about the modified live-attenuated vaccines pertain to their safety. In Danish swine herds, Ingelvac PRRS MLV vaccine virus has been shown to be capable of reverting to a pathogenic phenotype during replication in pigs. Additionally, Mengeling et al. confirmed that numerous vaccine-like field isolates, which contained the same restriction site marker that is found in the Ingelvac PRRS MLV vaccine virus, were capable of causing disease more severe than any clinical signs induced by the MLV. The restriction site marker was not identified in any isolates collected prior to the introduction of the Ingelvac PRRS MLV vaccine except for the parent strain ATCC-VR2332. Other vaccine-like strains have also been reported (9, 16, 23) and some of these isolates were shown to be mildly to moderately pathogenic in pigs. (23).
Due to the widespread use of Ingelvac® PRRS MLV and the periodic identification of vaccine-like isolates, there is a demand for a rapid assay that can be used routinely for identifying and differentiating these vaccine-like isolates from field isolates of PRRSV. Current methods for differentiating PRRSV isolates include PCR amplification, subsequent sequencing and sequence analyses, or PCR-restriction fragment length polymorphism. (27). Because these assays are costly and time-consuming, they are not very well suited for routine large-scale screening of viruses.
The heteroduplex mobility assay (HMA) is a rapid and inexpensive method of differentiating viral isolates. Delwart et al. (6) originally developed the assay for genetic typing of human immunodeficiency virus. More recently, HMA techniques have been applied to the study of other viruses such as influenza virus (29), feline immunodeficiency virus (2), measles virus (10), poliovirus (5), Newcastle disease virus (3), and hepatitis C virus (7, 28). The assay relies on the formation of mismatched base pairs when two closely related DNA molecules are combined, denatured, and reannealed. The mismatches cause structural distortions in the newly formed DNA molecule, resulting in heteroduplexes with reduced mobility on a polyacrylamide gel. It has been shown that the reduction in mobility is proportional to the degree of divergence between the two sequences. (6). The HMA, however, suffers from some of the disadvantages inherent in all screening techniques. Screening techniques assess the diversity of a sequence mixture based on indirect detection of sequence variations. Thus, these methods typically underestimate the true diversity of a sequence mixture.
As already noted, because of the high mutation rate of PRRSV and incidence of MLV vaccine revertant, a means of differentiating infected from vaccinated animals (DIVA) test would be desirable.
In furthering the industry's focus in potentially eradicating PRRS, a potential strategy involves developing marker vaccines and accompanying differential diagnostic tests. Currently, no such strategies exist in the industry for PRRS that are effective. While there is a commercial ELISA available for detecting antibody against PRRS virus, this commercial ELISA cannot differentiate vaccine-induced antibody from natural infection.
It is therefore a primary objective of the present invention to provide a more definitive strategy for PRRSV serological diagnosis.
It is a further objective of the present invention to provide a serological method of differentiating pigs naturally infected with PRRSV from animals vaccinated against PRRS.
It is still a further objective of the present invention to provide a means of differentiating pigs naturally infected with PRRSV from animals vaccinated against PRRS using the N-terminal region of 2b protein from PRRSV.
It is yet a further objective of the present invention to provide a means of differentiating pigs naturally infected with PRRSV from animals vaccinated against PRRS using an ELISA.
These and other objectives will become clear from the following detailed description of the invention.